1. To investigate NRP ligand binding sites. We have determined that VEGF-A, PlGF and heparin bind to the blb2 extracellular domain (ECD). VEGF-B and plexin A (which transduces the NRP signal in response to semaphorins) also bind to NRP ECD but the binding domains for these two proteins are unknown. Future studies include i) determining the VEGF-B and plexin-A binding sites on NRP using the myc-tagged domain pull-down strategy that previously determined the VEGF-A and PlGF binding sites on NRPs (Mamluk et al., JBC, 2002) and determining whether these ligand binding sites are unique or overlapping by analysis of deletion mutants and site-directed mutagenesis; ii) structural analysis of the heparin that binds NRP blb2 in collaboration with Dr John Gallagher (University of Manchester, UK); iii) crystallizing blb2, with and without VEGF165 and with heparin in collaboration with Dr. Celia Hamson (BBRI, Boston), who previously has crystallized Ephrin B2 (see letter). 2. To analyze regulation of NRP expression. Little is known about how NRP expression is regulated and whether NRPl and NRP2 are regulated differentially. We have isolated the NRPl and NRP2 promoters and have made NRP-luciferase constructs. We have found so far that phorbol ester, EGF and HB-EGF induce NRPl promoter activity in EC, tumor cells and keratinocytes. Future studies include: i) using NRP promoter-reporter constructs to screen inducers and inhibitors of NRP expression, and identifylng the transcription factors involved; ii) analysis of NRPl and NRP2 expression in the transition of vascular to lymphatic EC upon prox-Z overexpression (see letter from Dr. Michael Detmar); iii) analysis of comparative NRP and sNRP expression in cells, tissues and mouse embryos; iv) developmental and adult NRP expression patterns in NRPlLacZ and NRP2LacZ mice generated by Dr. Seiji Takashima, a former post-doc now in Osaka, Japan. 3. To analyze the non-neuronal properties of semaphorins. Semaphorins regulate axon guidance via NRPs by repelling axons and collapsing growth cones. We have found that semaphorins bind non-neuronal cells, e.g., EC, tumor cells and keratinocytes but the consequences of these interactions require further analysts. Future studies include: i) generating large amounts of Sema3A and 3F protein from cell cultures since semaphorins are not commercially available due in part to lack of stability; ii) determining whether Sema3NSema3F affect EC and tumor cell motility, proliferation, survival and apoptosis; iii) analyzing the effects of Sema3AL/3F on the actin cytoskeleton; iv) determining whether Sema 3A/ 3F are angiogenesis inhibitors. 4. To analyze NRP signaling pathways activated in response to VEGF165 and semaphorins. VEGF165 and semaphorins bind to NRPs expressed by a number of non-neuronal cell types. However, very little is known about how NRP signals in these cell types in response to these ligands. As a guide, a number of molecules have been implicated in semaphorin-induced growth cone collapse, including Plexin A1 and A2, the Rho family of GTPases, CRMP and GSK3. Actin filament reorganization is regulated by LIM kinase and its substrate, cofilin. Future studies include incubating EC, tumor cells and keratinocytes expressing NRPl/NRP2 with VEGF165, VEGFIZI (which doesn?t bind NRP as a control), VEGF-B and PlGF (which bind NRPl), VEGF-C (which binds NRP2), Sema3A (which binds NRP1) and Sema3F (which binds NRP2), and monitoring downstream effects by i) direct examination of signaling molecules implicated in growth cone collapse as described above; ii) western. blot with antiphosphotyrosine, serine and threonine antibodies; iii) 2D gel/proteomics to detect changes in general protein and phosphoproteins profiles; iv) transcriptional profiling of these treatments by microarray. It will be determined whether VEGF165 and semaphorins use similar or different pathways. Effects on cytoskeleton will be analyzed in collaboration with Dr. Don Ingber in our department, a well known expert in this area (see letter). Another approach is to inhibit NRP expression with antisense (RNAi, morpholinos) and analyze downstream effects. 5. To analyze the function of naturally occurring soluble NRP (sNRP). sNRP binds VEGF165 and is a VEGF165 antagonist. Future studies include: i) analyzing the effects of sNRPl on VEGF165-induced migration, proliferation and survival and determining the mechanisms involved e.g., inhibition of phosphorylation of specific tyrosine residues and of factors involved in migration such as PI3 kinase and AKT; ii) analyzing effects of sNW1 on angiogenesis and vascular permeability (VPF) activity in transgenic mice overexpressing sNRP in the skin using a K14 promoter, generated in collaboration with Dr. Michael Detmar, MGH (see letter); iii) analyzing mechanisms by which sNRP inhibits tumor progression, for example by inducing tumor cell apoptosis. 6. To investigate the role of NRPs in tumor angiogenesis and cancer. Overexpression of NWli enhances tumor angiogenesis and tumor growth in rodent models. Future experiments include i) overexpression of-NRP2 in human tumor cell lines (e.g., melanoma, squamous carcinoma); ii) overexpression of NRP antagonists, e.g., sNRP and semaphorins in human tumor cell lines. 7. To develop a proteomics program including optimizing phosphoprotein analysis to study signding. The Specific Aims of the original proposal were: 1. To Investigate VEGF165 and Semaphorid Collapsin Interactions with NRPl including: a,) mechanisms of VEGFI~S/~RPI/KDinRte ractions in EC; b) semaphorins as antagonists of EC and tumor cell motility via NRPI; and c) whether VEGF165 and semaphorins are competitive inhibitors of each other for interactions with EC and tumor cells. 2. To Investigate NRPl Expression and Function in Tumor Cells including: a) whether VEGFIbj is a direct stimulator of tumor cell motility acting via NRPl and what pathways may be involved; b) NRPl expression in tumor cells and the contribution of NRPl expression to tumor cell motility and metastatic potential. 3. To Characterize the Structure, Function and Distribution of a Naturally-Occurring Soluble NRPl (sNRP1) Receptor including: a) cloning and purification of a naturally occurring soluble NRPI; b) sNRPl as an antagonist of VEGFlbj-induced cell motility, proliferation and angiogenesis; c) the distribution of sNRP1 in comparison to intact membrane-bound NRPl; d) soluble receptor assays in vitro for analyzing modulators of VEGFlsNRPl interactions. 4. To Investigate the Regulation of NRPl Gene Expression and Activity including: a) cloning the NRPI and NRP2 promoters and identifying regulators of NRP gene expression; b) identifying cytoplasmic proteins that interact with the NRPI cytoplasmic domain using the yeast two hybrid system; c) characterization of a PDZ domain-containing protein.